A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines

Syombua, Easter D.; Adero, Mark Ochieng; Mbinda, Wilton; Wanyonyi, Christine N.; Ngugi, Mathew Piero; Oduor, Richard

doi:10.1007/s11816-020-00654-1

Cited by 4 publications

(4 citation statements)

References 39 publications

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“…In the present study, cassava regeneration frequency and transformation efficiency were low, in the range of 5.91-6.82% and 1.33-2%, respectively (Supplementary Materials 4 and 5), respectively. Syombua et al (2021) also reported a similar transformation frequency of 0.5% for TMS 60444 cultivar. The low efficiency observed could be explained by the selective stress of calli caused by hygromycin and timentin antibiotics.…”

Section: Discussionmentioning

confidence: 61%

“…Agrobacterium -mediated transformation and somatic embryogenesis were performed as previously described by Odipio et al. (2017) and Syombua et al. (2021) .…”

Section: Resultsmentioning

confidence: 99%

“…The gRNA for CRISPR/Cas9 editing was synthesized u s i n g t h e t a r g e t s e q u e n c e hi g hl ig h t e d i n y e l l o w (Supplementary Material 2). Agrobacterium-mediated transformation and somatic embryogenesis were performed as previously described by Odipio et al (2017) and Syombua et al (2021). After repeatedly subculturing the tissue and performing antibiotic selection on a selective medium (Figures 1A-C), multiple hygromycin-resistant cotyledon-stage embryos were generated from original leaf explants (Figures 1D, E).…”

Section: Cas9/grna Agrobacterium-mediated Transformationmentioning

confidence: 99%

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Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava

et al. 2022

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Cassava is the world’s most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.

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Section: Discussionmentioning

confidence: 61%

“…Agrobacterium -mediated transformation and somatic embryogenesis were performed as previously described by Odipio et al. (2017) and Syombua et al. (2021) .…”

Section: Resultsmentioning

confidence: 99%

Section: Cas9/grna Agrobacterium-mediated Transformationmentioning

confidence: 99%

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Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava

et al. 2022

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“…Explants have also been developed from the hypocotyls and cotyledons of the initial zygotic embryo and the apical buds, young leaf lobes, axillary buds, flower tissues, and cotyledons of primary, secondary, and cyclic somatic embryos. The development of somatic embryogenesis and plant regeneration laid a foundation for the induction of friable embryogenic callus (FEC) [23] and genetic transformation in cassava [24].…”

Section: Somatic Embryogenesismentioning

confidence: 99%

Plant Regeneration from Cassava Protoplasts

Wen¹,

Fu²,

Luo³

et al. 2024

Cassava - Recent Updates on Food, Feed, and Industry

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Cassava is an important crop for food, feed, and industrial raw materials. Given that traditional conventional breeding is restricted by various factors, biotechnology breeding has become an important breeding method. Tissue culture regeneration is the basis of biotechnology breeding. This chapter reviews the establishment and development of cassava tissue culture and regeneration systems and the technical processes of tissue culture and regeneration starting from the induction of explants of tissue-cultured cassava plantlets to embryogenic calli, isolation to protoplasts, culture to embryogenic calli followed by differentiation into embryos, and then sprouting, stemming, and rooting into complete plants. This chapter focuses on the technical processes from protoplast to complete plant and summarizes the important influencing factors of protoplast regeneration, which is the key and difficult point in the entire regeneration process of cassava protoplasts. This chapter aims to provide technical guidance for cassava protoplast regeneration, offer useful inspiration and reference for cassava tissue culture, and lay a foundation for the genetic improvement of cassava.

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Micropropagation of Cassava (Manihot esculenta Crantz): Review

Feyisa¹

2021

Extsv. Rev.

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Cassava is a vital crop to the food security of millions of people worldwide, particularly in Sub-Saharan Africa. Because the crop produced a reasonable yield on marginal soils, it could help relieve global hunger. As a result, increasing cassava output and its quality attributes are significant. However, the low multiplication rate of this main crop has resulted in the delayed dissemination of improved varieties among farmers. As a result, tissue culture techniques may be a feasible solution for overcoming these challenges. Cassava in vitro propagation had done using either the shoots multiplication technique or somatic embryogenesis. However, the shoot multiplication approach is preferable since it retains clonal fidelity. Plant regeneration via somatic embryogenesis or organogenesis entailed the use of numerous basal media containing various plant growth hormones. Several studies found that each type of cassava clone required a unique protocol to achieve optimal shoot initiation, shoot multiplication, root induction, and elongation. This review describes recent research on cassava micropropagation that makes use of a variety of experimental systems. While each of these systems focuses on a different aspect of technique, they can be significant in understanding the in vitro production of cassava planting material.

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A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines

Cited by 4 publications

References 39 publications

Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava

Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava

Plant Regeneration from Cassava Protoplasts

Micropropagation of Cassava (Manihot esculenta Crantz): Review

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